Summary While glial research has advanced in rodent models, significantly less progress has been made in understanding human-specific diversity of glia at a molecular and a functional level, both during development and in adulthood. To this end, our lab has led an active effort for the past several years to develop novel methodologies that isolate glial populations from normal and pathological human brain tissue, capturing more accurately the cells' native niche and molecular imprint and preserving their viability for subsequent functional analyses [1-4]. Recently, we also developed astrocyte-specific and oligodendroglial progenitor (OPC)-specific immunotagging strategy in fresh-frozen brain, enabling us to isolate nuclear RNA and chromatin from our large cohort of developing and adult frozen human brain collection with short postmortem/ischemia time interval. The goal of this study is to generate more accurate and detailed epigenetic and functional reference datasets for developing and adult human astrocyte and oligodendroglial lineages, and to make them readily available to the community for further disease-oriented human studies. Astrocytes and OPCs (also known as NG2+ glia) are two developmentally related but functionally distinct glial subtypes, the molecular signatures of which remain poorly defined in normal and pathological human conditions. Both lineages arise from a common precursor and mature into distinct phenotypes, but retain functional plasticity for proliferation in response to injury. We hypothesized that such functional plasticity is imprinted during development and reactivated in adulthood, through lineage-specific chromatin accessibility and transcriptional regulation. Leveraging our novel isolation tools, unique tissue collection, neuroanatomical skills, the lab's expertise in glial development and molecular neuroepigenetics, and our collaborators' expertise with computational single cell analyses, this proposal aims to elucidate further the relationship between functional and molecular plasticity in human astrocyte and OPC lineages during development, by employing high throughput technology to characterize their dynamic chromatin structure (aim1), transcription factor (TF) binding activity (aim2), and growth and differentiation kinetics (aim3). We will generate ATAC-seq, RNA-seq, ChIP-seq, and Tn5-HiC datasets for astrocyte and OPC lineages across four developmental time points and adulthood, isolated from germinal matrix and cortical plate niches, and will integrate bulk and single nucleus data in our analyses. The outlined molecular and functional experiments will enable us to create an integrated dynamic map of glial plasticity, offering a much needed reference toolset for further comparative studies.